Apply a score cutoff to filter variants based on a recalibration table

Category Variant Filtering

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Overview

Apply a score cutoff to filter variants based on a recalibration table

This tool performs the second pass in a two-stage process called Variant Quality Score Recalibration (VQSR). Specifically, it applies filtering to the input variants based on the recalibration table produced in the first step by VariantRecalibrator and a target sensitivity value, which the tool matches internally to a VQSLOD score cutoff based on the model's estimated sensitivity to a set of true variants.

The filter determination is not just a pass/fail process. The tool evaluates for each variant which "tranche", or slice of the dataset, it falls into in terms of sensitivity to the truthset. Variants in tranches that fall below the specified truth sensitivity filter level have their FILTER field annotated with the corresponding tranche level. This results in a callset that is filtered to the desired level but retains the information necessary to increase sensitivity if needed.

To be clear, please note that by "filtered", we mean that variants failing the requested tranche cutoff are marked as filtered in the output VCF; they are not discarded unless the option to do so is specified.

Summary of the VQSR procedure

The purpose of variant recalibration is to assign a well-calibrated probability to each variant call in a call set. These probabilities can then be used to filter the variants with a greater level of accuracy and flexibility than can typically be achieved by traditional hard-filter (filtering on individual annotation value thresholds). The first pass consists of building a model that describes how variant annotation values co-vary with the truthfulness of variant calls in a training set, and then scoring all input variants according to the model. The second pass simply consists of specifying a target sensitivity value (which corresponds to an empirical VQSLOD cutoff) and applying filters to each variant call according to their ranking. The result is a VCF file in which variants have been assigned a score and filter status.

VQSR is probably the hardest part of the Best Practices to get right, so be sure to read the method documentation, parameter recommendations and tutorial to really understand what these tools do and how to use them for best results on your own data.

Inputs

• The raw input variants to be filtered.
• The recalibration table file that was generated by the VariantRecalibrator tool.
• The tranches file that was generated by the VariantRecalibrator tool.

Output

• A recalibrated VCF file in which each variant of the requested type is annotated with its VQSLOD and marked as filtered if the score is below the desired quality level.

Usage examples

Applying recalibration/filtering to SNPs

 gatk ApplyVQSR \
   -R Homo_sapiens_assembly38.fasta \
   -V input.vcf.gz \
   -O output.vcf.gz \
   --ts_filter_level 99.0 \
   --tranches-file output.tranches \
   --recal-file output.recal \
   -mode SNP
 

Allele-specific version of the SNP filtering (beta)

 gatk ApplyVQSR \
   -R Homo_sapiens_assembly38.fasta \
   -V input.vcf.gz \
   -O output.vcf.gz \
   -AS \
   --ts_filter_level 99.0 \
   --tranches-file output.AS.tranches \
   --recal-file output.AS.recal \
   -mode SNP 
 

Note that the tranches and recalibration files must have been produced by an allele-specific run of VariantRecalibrator. Also note that the AS_culprit, AS_FilterStatus, and AS_VQSLOD fields will have placeholder values (NA or NaN) for alleles of a type that have not yet been processed by ApplyRecalibration. The spanning deletion allele (*) will not be recalibrated because it represents missing data. Its VQSLOD will remain NaN, and its culprit and FilterStatus will be NA.

Each allele will be annotated by its corresponding entry in the AS_FilterStatus INFO field annotation. Allele-specific VQSLOD and culprit are also carried through from VariantRecalibrator, and stored in the AS_VQSLOD and AS_culprit INFO fields, respectively. The site-level filter is set to the most lenient of any of the allele filters. That is, if one allele passes, the whole site will be PASS. If no alleles pass, the site-level filter will be set to the lowest sensitivity tranche among all the alleles.

Caveats

• The tranche values used in the example above are only meant to be a general example. You should determine the level of sensitivity that is appropriate for your specific project. Remember that higher sensitivity (more power to detect variants, yay!) comes at the cost of specificity (more false negatives, boo!). You have to choose at what point you want to set the tradeoff.
• In order to create the tranche reporting plots (which are only generated for SNPs, not indels!) the Rscript executable needs to be in your environment PATH (this is the scripting version of R, not the interactive version).

ApplyVQSR specific arguments

This table summarizes the command-line arguments that are specific to this tool. For more details on each argument, see the list further down below the table or click on an argument name to jump directly to that entry in the list.

Argument name(s)	Default value	Summary
Required Arguments
--output  -O	null	The output filtered and recalibrated VCF file in which each variant is annotated with its VQSLOD value
--recal-file	null	The input recal file used by ApplyRecalibration
--variant  -V	[]	One or more VCF files containing variants
Optional Tool Arguments
--arguments_file	[]	read one or more arguments files and add them to the command line
--cloud-index-prefetch-buffer  -CIPB	-1	Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.
--cloud-prefetch-buffer  -CPB	40	Size of the cloud-only prefetch buffer (in MB; 0 to disable).
--disable-bam-index-caching  -DBIC	false	If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.
--exclude-filtered	false	Don't output filtered loci after applying the recalibration
--gcs-max-retries  -gcs-retries	20	If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection
--help  -h	false	display the help message
--ignore-all-filters	false	If specified, the variant recalibrator will ignore all input filters. Useful to rerun the VQSR from a filtered output file.
--ignore-filter	[]	If specified, the recalibration will be applied to variants marked as filtered by the specified filter name in the input VCF file
--interval-merging-rule  -imr	ALL	Interval merging rule for abutting intervals
--intervals  -L	[]	One or more genomic intervals over which to operate
--mode	SNP	Recalibration mode to employ: 1.) SNP for recalibrating only SNPs (emitting indels untouched in the output VCF); 2.) INDEL for indels; and 3.) BOTH for recalibrating both SNPs and indels simultaneously.
--reference  -R	null	Reference sequence
--tranches-file	null	The input tranches file describing where to cut the data
--truth-sensitivity-filter-level  -ts-filter-level	null	The truth sensitivity level at which to start filtering
--use-allele-specific-annotations  -AS	false	If specified, the tool will attempt to apply a filter to each allele based on the input tranches and allele-specific .recal file.
--version	false	display the version number for this tool
Optional Common Arguments
--add-output-sam-program-record	true	If true, adds a PG tag to created SAM/BAM/CRAM files.
--add-output-vcf-command-line	true	If true, adds a command line header line to created VCF files.
--create-output-bam-index  -OBI	true	If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.
--create-output-bam-md5  -OBM	false	If true, create a MD5 digest for any BAM/SAM/CRAM file created
--create-output-variant-index  -OVI	true	If true, create a VCF index when writing a coordinate-sorted VCF file.
--create-output-variant-md5  -OVM	false	If true, create a a MD5 digest any VCF file created.
--disable-read-filter  -DF	[]	Read filters to be disabled before analysis
--disable-sequence-dictionary-validation	false	If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!
--disable-tool-default-read-filters	false	Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)
--exclude-intervals  -XL	[]	One or more genomic intervals to exclude from processing
--gatk-config-file	null	A configuration file to use with the GATK.
--input  -I	[]	BAM/SAM/CRAM file containing reads
--interval-exclusion-padding  -ixp	0	Amount of padding (in bp) to add to each interval you are excluding.
--interval-padding  -ip	0	Amount of padding (in bp) to add to each interval you are including.
--interval-set-rule  -isr	UNION	Set merging approach to use for combining interval inputs
--lenient  -LE	false	Lenient processing of VCF files
--QUIET	false	Whether to suppress job-summary info on System.err.
--read-filter  -RF	[]	Read filters to be applied before analysis
--read-index	[]	Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.
--read-validation-stringency  -VS	SILENT	Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.
--seconds-between-progress-updates	10.0	Output traversal statistics every time this many seconds elapse
--sequence-dictionary	null	Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.
--TMP_DIR	[]	Undocumented option
--use-jdk-deflater  -jdk-deflater	false	Whether to use the JdkDeflater (as opposed to IntelDeflater)
--use-jdk-inflater  -jdk-inflater	false	Whether to use the JdkInflater (as opposed to IntelInflater)
--verbosity	INFO	Control verbosity of logging.
Advanced Arguments
--lod-score-cutoff	null	The VQSLOD score below which to start filtering
--showHidden	false	display hidden arguments

Argument details

Arguments in this list are specific to this tool. Keep in mind that other arguments are available that are shared with other tools (e.g. command-line GATK arguments); see Inherited arguments above.

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--add-output-sam-program-record / -add-output-sam-program-record

If true, adds a PG tag to created SAM/BAM/CRAM files.

boolean  true

---

--add-output-vcf-command-line / -add-output-vcf-command-line

If true, adds a command line header line to created VCF files.

boolean  true

---

--arguments_file / NA

read one or more arguments files and add them to the command line

List[File]  []

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--cloud-index-prefetch-buffer / -CIPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable). Defaults to cloudPrefetchBuffer if unset.

int  -1  [ [ -∞  ∞ ] ]

---

--cloud-prefetch-buffer / -CPB

Size of the cloud-only prefetch buffer (in MB; 0 to disable).

int  40  [ [ -∞  ∞ ] ]

---

--create-output-bam-index / -OBI

If true, create a BAM/CRAM index when writing a coordinate-sorted BAM/CRAM file.

boolean  true

---

--create-output-bam-md5 / -OBM

If true, create a MD5 digest for any BAM/SAM/CRAM file created

boolean  false

---

--create-output-variant-index / -OVI

If true, create a VCF index when writing a coordinate-sorted VCF file.

boolean  true

---

--create-output-variant-md5 / -OVM

If true, create a a MD5 digest any VCF file created.

boolean  false

---

--disable-bam-index-caching / -DBIC

If true, don't cache bam indexes, this will reduce memory requirements but may harm performance if many intervals are specified. Caching is automatically disabled if there are no intervals specified.

boolean  false

---

--disable-read-filter / -DF

Read filters to be disabled before analysis

List[String]  []

---

--disable-sequence-dictionary-validation / -disable-sequence-dictionary-validation

If specified, do not check the sequence dictionaries from our inputs for compatibility. Use at your own risk!

boolean  false

---

--disable-tool-default-read-filters / -disable-tool-default-read-filters

Disable all tool default read filters (WARNING: many tools will not function correctly without their default read filters on)

boolean  false

---

--exclude-filtered / NA

Don't output filtered loci after applying the recalibration

boolean  false

---

--exclude-intervals / -XL

One or more genomic intervals to exclude from processing
Use this argument to exclude certain parts of the genome from the analysis (like -L, but the opposite). This argument can be specified multiple times. You can use samtools-style intervals either explicitly on the command line (e.g. -XL 1 or -XL 1:100-200) or by loading in a file containing a list of intervals (e.g. -XL myFile.intervals).

List[String]  []

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--gatk-config-file / NA

A configuration file to use with the GATK.

String  null

---

--gcs-max-retries / -gcs-retries

If the GCS bucket channel errors out, how many times it will attempt to re-initiate the connection

int  20  [ [ -∞  ∞ ] ]

---

--help / -h

display the help message

boolean  false

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--ignore-all-filters / NA

If specified, the variant recalibrator will ignore all input filters. Useful to rerun the VQSR from a filtered output file.

boolean  false

---

--ignore-filter / NA

If specified, the recalibration will be applied to variants marked as filtered by the specified filter name in the input VCF file
For this to work properly, the -ignoreFilter argument should also be applied to the VariantRecalibration command.

List[String]  []

---

--input / -I

BAM/SAM/CRAM file containing reads

List[String]  []

---

--interval-exclusion-padding / -ixp

Amount of padding (in bp) to add to each interval you are excluding.
Use this to add padding to the intervals specified using -XL. For example, '-XL 1:100' with a padding value of 20 would turn into '-XL 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]

---

--interval-merging-rule / -imr

Interval merging rule for abutting intervals
By default, the program merges abutting intervals (i.e. intervals that are directly side-by-side but do not actually overlap) into a single continuous interval. However you can change this behavior if you want them to be treated as separate intervals instead.

The --interval-merging-rule argument is an enumerated type (IntervalMergingRule), which can have one of the following values:

ALL

OVERLAPPING_ONLY

IntervalMergingRule  ALL

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--interval-padding / -ip

Amount of padding (in bp) to add to each interval you are including.
Use this to add padding to the intervals specified using -L. For example, '-L 1:100' with a padding value of 20 would turn into '-L 1:80-120'. This is typically used to add padding around targets when analyzing exomes.

int  0  [ [ -∞  ∞ ] ]

---

--interval-set-rule / -isr

Set merging approach to use for combining interval inputs
By default, the program will take the UNION of all intervals specified using -L and/or -XL. However, you can change this setting for -L, for example if you want to take the INTERSECTION of the sets instead. E.g. to perform the analysis only on chromosome 1 exomes, you could specify -L exomes.intervals -L 1 --interval-set-rule INTERSECTION. However, it is not possible to modify the merging approach for intervals passed using -XL (they will always be merged using UNION). Note that if you specify both -L and -XL, the -XL interval set will be subtracted from the -L interval set.

The --interval-set-rule argument is an enumerated type (IntervalSetRule), which can have one of the following values:

UNION

Take the union of all intervals

INTERSECTION

Take the intersection of intervals (the subset that overlaps all intervals specified)

IntervalSetRule  UNION

---

--intervals / -L

One or more genomic intervals over which to operate

List[String]  []

---

--lenient / -LE

Lenient processing of VCF files

boolean  false

---

--lod-score-cutoff / NA

The VQSLOD score below which to start filtering

Double  null

---

--mode / -mode

Recalibration mode to employ: 1.) SNP for recalibrating only SNPs (emitting indels untouched in the output VCF); 2.) INDEL for indels; and 3.) BOTH for recalibrating both SNPs and indels simultaneously.

The --mode argument is an enumerated type (Mode), which can have one of the following values:

SNP

INDEL

BOTH

Mode  SNP

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--output / -O

The output filtered and recalibrated VCF file in which each variant is annotated with its VQSLOD value

R String  null

---

--QUIET / NA

Whether to suppress job-summary info on System.err.

Boolean  false

---

--read-filter / -RF

Read filters to be applied before analysis

List[String]  []

---

--read-index / -read-index

Indices to use for the read inputs. If specified, an index must be provided for every read input and in the same order as the read inputs. If this argument is not specified, the path to the index for each input will be inferred automatically.

List[String]  []

---

--read-validation-stringency / -VS

Validation stringency for all SAM/BAM/CRAM/SRA files read by this program. The default stringency value SILENT can improve performance when processing a BAM file in which variable-length data (read, qualities, tags) do not otherwise need to be decoded.

The --read-validation-stringency argument is an enumerated type (ValidationStringency), which can have one of the following values:

STRICT

LENIENT

SILENT

ValidationStringency  SILENT

---

--recal-file / NA

The input recal file used by ApplyRecalibration

R FeatureInput[VariantContext]  null

---

--reference / -R

Reference sequence

String  null

---

--seconds-between-progress-updates / -seconds-between-progress-updates

Output traversal statistics every time this many seconds elapse

double  10.0  [ [ -∞  ∞ ] ]

---

--sequence-dictionary / -sequence-dictionary

Use the given sequence dictionary as the master/canonical sequence dictionary. Must be a .dict file.

String  null

---

--showHidden / -showHidden

display hidden arguments

boolean  false

---

--TMP_DIR / NA

Undocumented option

List[File]  []

---

--tranches-file / NA

The input tranches file describing where to cut the data

File  null

---

--truth-sensitivity-filter-level / -ts-filter-level

The truth sensitivity level at which to start filtering

Double  null

---

--use-allele-specific-annotations / -AS

If specified, the tool will attempt to apply a filter to each allele based on the input tranches and allele-specific .recal file.
Filter the input file based on allele-specific recalibration data. See tool docs for site-level and allele-level filtering details. Requires a .recal file produced using an allele-specific run of VariantRecalibrator.

boolean  false

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--use-jdk-deflater / -jdk-deflater

Whether to use the JdkDeflater (as opposed to IntelDeflater)

boolean  false

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--use-jdk-inflater / -jdk-inflater

Whether to use the JdkInflater (as opposed to IntelInflater)

boolean  false

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--variant / -V

One or more VCF files containing variants

R List[String]  []

---

--verbosity / -verbosity

Control verbosity of logging.

The --verbosity argument is an enumerated type (LogLevel), which can have one of the following values:

ERROR

WARNING

INFO

DEBUG

LogLevel  INFO

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--version / NA

display the version number for this tool

boolean  false

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GATK version 4.0.4.0 built at 02-47-2019 02:47:54.